Monitoring real-time pcr with label free intrinsic imaging

ABSTRACT

The invention provides a method for the detection of nucleic acid the method comprising carrying out a PCR reaction in a microfluidic device wherein the sample shuttles within the microfluidic channel and the amount of nucleic acid is determined based on the UV absorption of the nucleic acid.

BACKGROUND

The detection of nucleic acid and of nucleic acid modifications has manyapplications and is a particularly important tool in the diagnosis ofdisease. Most techniques employ the Polymerase Chain Reaction (PCR),which enables the amplification of very small amounts of complex geneticmaterial.

The advent of PCR has hugely accelerated the progress of studies on thegenetic structure of a diversity of organisms. PCR is anenzyme-catalysed reaction that allows any nucleic acid sequence to begenerated in vitro, and in abundance. First reported in 1986, PCR hassince become a requisite tool in basic molecular biology, genomesequencing, clinical research and evolutionary studies. The reason forthe success of PCR lies in its simplicity. At high temperature (about95° C.), the double-stranded target DNA denatures—unwinds into twosingle strands. Synthetic sequences of single-stranded DNA, known asprimers, are used to bracket the region of the chain to be amplified:one primer is complementary to one DNA strand (at the start of thetarget region), with the second primer being complementary to the otherDNA strand (at the end of the target region). The primers are annealedto the single strands when the local temperature is reduced to between50 and 65° C. This is followed by ‘extension’, at a slightly highertemperature (about 72° C.), in which a complementary strand developsfrom each primer by the catalytic action of a DNA polymerase enzyme, inthe presence of free deoxynucleotide triphosphates. This three-stepprocess constitutes one PCR cycle, and if repeated n times will yield 2″copies of the original DNA strand.

Conventional instruments for performing PCR (thermal cyclers) areconceptually simple, but possess a number of technical frailties thatlimit the speed and efficiency of amplification. A fundamentalrequirement for efficient amplification is rapid heat transfer. It isdesirable to have a system with a low heat capacity that can transferheat quickly to the sample on heating, and quickly away when cooling.Most conventional thermal cyclers have large thermal masses, resultingin high power requirements and protracted heating and cooling rates.Consequently, total reaction times are typically in excess of 90minutes. As the denaturation and annealing steps occur as soon as thecorrect temperature is reached, and extension is limited only by theprocessing power of the polymerase enzyme (between 50 and 500 bases persecond), total reaction times can be drastically shortened if thethermal mass of the instrument is reduced. In recent years,microfabricated PCR systems have been developed with this idea in mind:although diverse in structure, all rely on the reduction of thermal massto facilitate rapid heating and cooling, and afford reaction times asshort as a few minutes. The normal approach to instrumentminiaturisation involves the direct downsizing of system dimensions toreduce thermal masses. Early studies used this concept to createmicrofabricated devices in which a static reaction chamber (with volumesbetween 100 pL and 50 μL) was thermally cycled using resistive heatersmounted externally or integrated within a monolithic substrate. Usingthis general approach significant improvement in reaction speed,analytical throughput and reaction efficiency have been demonstrated.

Traditional PCR methods analyse the product by agarose gelelectrophoresis. The disadvantage is that this method is time consumingand shows low sensitivity. The basic PCR method has been developedfurther and methods are now available for detecting sequence-specificPCR products in real time. One such method is the TaqMan® assay(Holland. P. et al 1991. PNAS, 88 pp 7276-7280 Detection of specificpolymerase chain reaction product by utilizing the 5′-->3′ exonucleaseactivity of Thermus aquaticus DNA polymerase.) wherein detection of PCRproducts is based on the detection of fluorescence of a reporter.However, despite its advantages, the TaqMan® assay also has somedisadvantages. For example, sequence data for the construction of probesmust be available. Therefore, the costs for the assay are particularlyhigh when different probes need to be synthesised for the detection ofdifferent sequences. Another method for real-time PCR commonly usedemploys a dye (SYBR Green®, Lipsky, R. et al 2001 Clinical Chemistry 47pp 635-644 DNA Melting Analysis for Detection of Single NucleotidePolymorphisms), which binds specifically to double-stranded DNA, but notto single-stranded DNA. However, this method has the disadvantage thatthe dye is non-specific and can generate false positive signals. Othermethods use molecular beacons or scorpions but similar to the TaqMan®assay, these methods are complex and expensive.

WO 03/102238 relates to a real-time PCR method by measuring UVabsorbance of the PCR mixture. The contents of WO 03/102238 are herebyincorporated by reference. WO 03/036302 discloses a method formonitoring the folding and unfolding of proteins and an apparatus foranalysing temperature-dependent configurations of proteins. The contentsof WO 03/036302 are hereby also incorporated by reference.

Due to genetic variations, more than 90% of drugs provide effectivetreatment in only 30%-50% of a given population. In order to reduce thisserious problem the healthcare industry is looking towards a morepersonal treatment regime. Currently, this age of personalised medicineis being delayed by the lack of diagnostic technology available at thePoint-of-Care (PoC). Nucleic acid modifications include short tandemrepeats (STR) and single nucleotide polymorphisms (SNPs). SNPs are DNAsequence variations that occur when a single nucleotide (A, T, C or G)in the genome sequence is altered. For a variation to be considered aSNP, it must occur in at least 1% of the population. Allele frequenciesvary greatly, also amongst different populations. SNPs, which make upabout 90% of all human genetic variation, occur every 100 to 300 basesalong the 3-billion-base human genome. Because SNPs are usually onlypresent in two forms, the allele that is more rare is referred to asmutant or minor allele and the most common allele is referred to as wildtype allele. SNPs are primarily bi-allelic (i.e. there are two possiblealleles at one locus) but may also be tri-allelic (i.e. two independentmutation events have occurred at the same time). Two of every three SNPsinvolve the replacement of cytosine (C) with thymine (T). SNPs can occurin both coding (gene) and non-coding regions of the genome (extronic orintronic).

Although more than 99% of human DNA sequences are the same across thepopulation, variations in DNA sequence can have a major impact on howhumans respond to disease, pathogens and therapies. This makes SNPs ofgreat value for biomedical research and for developing pharmaceuticalproducts or medical diagnostics. Therefore, the provision of anefficient, precise, cheap and user-friendly method for the detection ofSNPs can be of great value. Current methods used to analyse SNPs includePCR followed by sequencing, microarrays and mass spectrometry. However,in particular microarrays and mass spectrometry are complex andexpensive. One PCR method based on 3′ mismatch SNP scoring isexemplified by the GALIOS system (Weber, S. et al 2002 British Journalof Haematology 116 pp 839-843: Genotyping of human platelet antigen-1 bygene amplification and labeling in one system and automated fluorescencecorrelation spectroscopy). In this system two primers are used at the 5′end of the putative product, one of which has a 3′ mismatch. The upperwild type template has a complementary primer, which the polymerase willbe able to extend to the 3′. The lower SNP containing template cannotfully anneal the primer, creating a 3′ mismatch. This will result inmarkedly lower product, if indeed any. In GALIOS, and other relatedsystems, product is detected by attached fluorescent dyes. Otherlabelled systems also exist such as Taqman® and SYBR green systems andtheir disadvantages are discussed above. Therefore, there is a need foran alternative and improved method for analysing SNPs.

Most DNA molecules show a relative increase of 1.4 in absorbance at 260nm upon denaturation which is known as the hyperchromic effect. Thiseffect arises due to the different structures of single (ssDNA) anddouble stranded DNA (dsDNA). ssDNA absorbs about 30% more photons at 260nm than dsDNA. The increase in absorbance is caused by the exposure ofthe highly absorbing purine and pyrimidine rings, which in dsDNA arestacked internally to the helix, as they form the hydrogen bondmoieties.

Microfluidic devices have been described elsewhere (Kopp et al, Science280. 1998). For example, Münchow et al discloses a microfluidic PCRmethod wherein the PCR product is detected by fluorescence or gelelectrophoresis (Münchow G et al 2005 Expert Rev. Mol. Diagn. 5 pp613-620 Automated chip-based device for simple and fast nucleic acidamplification).

The present inventors have found an alternative method for nucleic aciddetection which makes use of a semi continuous flow PCR and is based onlabel free intrinsic imaging, as no extrinsic label needs to beincorporated into the PCR product.

The invention also provides a method for allele specific primer, PCRbased, SNP validation. Creation of such a method should allow ahealthcare worker to take a blood sample from a patient and rapidly(within a few minutes) make an informed choice of drug therapy based onthe patient's genetic information.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method for the detectionof nucleic acid the method comprising

-   -   a) amplifying a nucleic acid sample wherein a solution        comprising a nucleic acid sample is moved along a        temperature-controlled and UV illuminated channel and the flow        direction of the solution is altered multiple times and    -   b) measuring UV absorption of the nucleic acid.

In another aspect, the invention relates to an apparatus for determiningthe presence of nucleic acid comprising at least one microchannel, aferrofluidic actuation means, heating elements, a UV light source and adetector.

DESCRIPTION OF THE INVENTION

According to a first aspect, the invention relates to a method for thedetection of nucleic acid the method comprising

-   -   a) amplifying a nucleic acid sample wherein a solution        comprising a nucleic acid sample is moved along a        temperature-controlled and UV illuminated channel and the flow        direction of the solution is altered multiple times and    -   b) measuring UV absorption of the nucleic acid.

Thus, according to the invention, the solution comprising the nucleicacid sample moves backwards and forwards within the channel. Therefore,the solution moves in a non-linear and non-continuous fashion. Thesample shuttles between different parts of the channel.

The amplification is carried out while the solution shuttles. This isachieved by applying different temperatures to different parts of thechannel.

The method allows determining the amount of nucleic acid present in thesample.

According to the invention, the term nucleic acid sample refers to asample comprising DNA or RNA. The DNA may comprise cDNA and RNA maycomprise mRNA or siRNA. Preferably, the nucleic acid sample comprisessingle-stranded DNA or RNA or double-stranded DNA or RNA. In oneembodiment, the nucleic acid comprises native secondary structuralelements or is in its denatured form. In one embodiment, the nucleicacid sample may comprise nucleic acid isolated from a microorganism,animal or plant. In another embodiment, the nucleic acid is a syntheticsequence, for example a part of a vector or an oligonucleotide. In afurther embodiment, the nucleic acid sample comprises animal or plantcells or cells of a microorganism.

In one embodiment, the amplification is by PCR. According to theinvention, PCR refers to a polymerase chain reaction for theamplification of DNA. Typically, a PCR reaction comprises the steps ofdenaturation, annealing and extension, which are carried out atdifferent temperatures. Denaturation, the separation of twocomplementary strands, typically requires a standard temperature ofabout 95° C. The temperature required for annealing is dependent on theparticular primer used but is typically carried out at about 54° C., butvaries depending on the base composition of the primer. It may bebetween 40° C. and 60° C. Extension of primer molecules is typicallycarried out at about 72° C. A skilled person will appreciate that thetemperatures used vary according to the type of PCR carried out and theprimers used. PCR requires the presence of a polymerase enzyme tocatalyse the reaction. Typically, DNA Pol I, Taq polymerase or any otherthermally stable polymerase enzyme may be used.

PCR may be carried out in real time.

A primer is a short oligonucleotide which anneals to the complementarysequence within the target nucleic acid. Typically the primer is 15 to30 nucleotides long. The sequence of oligonucleotide primers used in thereaction is dependent on the sequence of interest. If required,degenerate primers may be used in the method of the invention. Theprecise temperature control of the channel enables to accurately adjustthe temperature required for the specific primer used.

In one embodiment method of the invention, the primer may also belabelled to provide a further level of detection. Such labels are knownto the skilled person and include, fluorescent dyes or radioactivelabels.

Thus, in one embodiment, the solution further comprises a PCR mixture.The term PCR mixture or PCR reagents according to the invention refersto a mixture comprising components typically required to perform a PCRreaction. Such mixture will typically comprise a buffer, a set of atleast one oligonucleotide primer, dNTP's (Nucleotides consisting of thefour DNA bases adenine (dATP), thymine (dTTP), guanine (dGTP) andcytosine (dCTP)), and a polymerase. It will be apparent to the skilledperson that the components of the PCR mixture and their concentrationand the conditions used may be varied according to the type of PCRreaction performed.

The skilled person will be aware that the method according to theinvention may relate to different types of PCR reactions, such as hotstart PCR, inverse PCR, RT-PCR, RACE, nested PCR, asymmetric PCR andother PCR methods. Thus the invention also relates to a method accordingto the invention wherein hot start PCR, inverse PCR, RT PCR, RACE,nested PCR, asymmetric PCR is carried out.

The term temperature-controlled according to the invention refers tocontrolling the temperature along the length of the channel, in otherwords, a temperature profile is applied to the length of the channel andthus to the solution within the channel. According to the invention, thetemperature is controlled so that a range of temperatures can be appliedto the solution along the length of the channel. Thus, a specifiedtemperature can be applied to the channel (and thus the solution) at anygiven poirit along the length of a channel. Thus, it is possible toapply a specific temperature to the solution at each possible positionalong the length of the channel. The channel may comprises Peltier cellsas temperature controlling elements. Temperature resolution can beadjusted to suit experimental conditions, but resolution of 1° C. permillimetre or lower may be used. In one embodiment, the channel is amicrochannel, for example on a chip, such as described in Kopp et al,Science 280, 1998.

In one embodiment, the temperature of the channel is within the range of40° C. to 110° C.

In another embodiment, different temperatures zones are applied toseparate parts of the channel. Preferably, the temperature in the firsttemperature zone is within the range of 40° C. to 60° C., in the secondtemperature zone about 72° C. and in the third temperature zone about95° C.

According to the invention, amplification is achieved by shunting asolution comprising a DNA sample and PCR reagents (“sample plug”) backand forth, over static heating zones, in a microfluidic channel, forexample by applying alternate left-right pressure as shown in FIG. 2(Münchow et al). The method combines the cycling flexibility of staticor well-based reactors with the rapid temperature transitions (andultra-low thermal masses) associated with continuous-flow PCRmicrostructure. In contrast to continuous flow PCR using microfluidicmethods known in the art, the method of the invention thus provides asemi-continuous flow PCR combined with the detection of nucleic acid onthe basis of measuring UV absorption of the nucleic acid.

According to the methods of the invention, information on the efficiencyof PCR is acquired from data relating to the hyperchromic effect (FIG.4). Thus, UV absorption of the nucleic acid sample is measured at about260 nm and the production of nucleic acid during the amplification canbe monitored.

Imaging the product plug during the denaturation phase at 95° C. may beenhanced by this feature. It may be envisioned that the increase indsDNA product will not be represented by a smooth exponential curve,rather it is expected to have features related to the hyperchromiceffect, resulting in extra absorbance generated as the DNA denatures.FIG. 5 shows a representation of how the product curve may appear.

A series of step features will be generated as the amount of productincreases. This new dsDNA product in the 72° C. zone will increase theabsorbance at 260 nm as it is created. As the reaction plug shuttles tothe 95° C. zone it will melt to ssDNA and show a signal increase due toboth the amount of product and the hyperchromic effect. As it shuttlesback to the 72° C. there will be a small initial drop in absorbance dueto the re-naturing of the strands, however this will soon be compensatedfor by the increase in dsDNA. This is one of a number of possibleoutcomes. It may be that no step features will occur, or that theincrease at 95° C. will produce a much sharper angle of product curve.It is also important to consider that the information gained will bedependent on the speed of data sampling. Imaging systems may operate atfrequencies of 20 Hertz (Hz) and higher across the 512 pixels of thePhoto Diode array or elements of a Charged Coupled Device (CCD).However, as the clock speed used is much higher and consequentlyultrafast (10 s of microsecond), imaging is also possible and can bemade to match the system chemistry dynamics. Multiple detectors may beused, and the speed of the shuttling plug controlled very accurately sothat the system will be tunable and can acquire the best data possiblewithin the denaturing zone.

The concentration or amount of the PCR product is monitored based onmeasuring UV absorption. Concentration is detected by causing thenucleic acid to pass between a light source and a light detector. Forexample, by using UV sensitised Photo Diode Arrays (PDAs) orcharge-coupled devices (CCDs) as detectors, the speed at which the DNAband moves across the channel can be determined, thus giving a measureof the plug length of the sample.

In one embodiment, the method comprises measuring the velocity of thenucleic acid in the sample. The velocity of the molecule can beestablished by the use of multiple detection as the molecule traversesone or more photo diode arrays of 512 or more pixels. This allows aspace-time correlation to be established. The position of the moleculewithin the channel is detected based on the UV absorption of the nucleicacid. The molecules are illuminated by Ultra Violet light from adeuterium lamp or UV diode laser which they maximally absorb at 260 nm,causing a drop in signal at the pixel of the PDA detector they aretraversing. Thus, the nucleic acid may be identified by signal which canbe used to obtain a measure of the amount present. It is also possibleto calculate the velocity that is needed for the sample to reach apredetermined position and based on the velocity of the sample, theamount present can be determined. The techniques used according to theinvention for velocity based signal processing are described inWO96/35946, WO 02/12876 and WO 02/12877. Multipixel detection alsoyields an increase in single-to-noise ratio when appropriate space-timeaveraging is performed. Placing a PDA close to the ends of the channel,and close to the temperature zones, will allow monitoring of anytemperature dependent features exhibited by the DNA. The exactconfiguration of the detectors, their number and their position will bea function of the imaging constraints introduced by the speed and sizeof the PCR plug. The sample plugs is positioned at t=0 before it beginsto move. This allows the application of velocity based signalprocessing. In the proposed system t=0 will be relatively easy toestablish as the plug will be stationary in the system as a function ofthe temperature zone it is in. In one configuration, the detectors maybe positioned contiguous to the heating element, or orthogonally.

Thus, in one embodiment, the method of the invention comprises velocitybased signal processing.

One of the advantages of the proposed method is that it allows real-timemeasurements to be performed. The number of PCR cycles needed forsatisfactory amplification using conventional PCR techniques is at least30. The method of the invention can significantly reduce the numbers ofcycles need to produce a detectable amount of product. As few as 10cycles of a typical 300 bp PCR product have been imaged using LFIItechnology (FIG. 7). According to the invention, PCR is carried outusing 10 to 40, preferably 20 cycles. The amount of times that the flowdirection of the solution comprising the sample and the PCR mixture, inother words the PCR sample plug, is altered thus depends on the cyclesof the PCR used.

Furthermore, even with the best of the PCR machines in general use, aPCR reaction will take at least an hour. More expensive machines can doit in less time, but they are not in general laboratory use. Accordingto the method of the invention, the reaction time can be reducedsignificantly.

Furthermore, the method requires low sample and reagent volumestypically, but not limited to the nL range. Due to the low instantaneousvolumes and associated thermal masses, sample plugs will thermallyequilibrate on a time scale of some between 10 and 100 ms whentransported to a different temperature zone. Consequently, the thermallimitations on the cycle speed of the system are greatly reduced whencompared with both macro- and microfluidic batch cyclers. Moreover,since PCR reagents will be contained within a relatively shortmicrochannel (when compared with a continuous-flow system) there is lessadsorption of vital reaction components such as the DNA polymerase) ontochannel surfaces.

The microfluidic channel used in the methods of the invention may bemade using current state of the art microfluidic techniques such as SU-8photolithograghy, and polydimethylsilaxane (PDMS), TOPAS or other UVtransparent plastic chip microfabrication, laser or machine tool or wetetching of a plurality of UV transparent glasses or quartz materials.

The method may use either pressure or ferro-fluidic actuation tomanipulate the flow direction of the solution within the channel. In thecase of ferro-fluidic actuation, the solution further comprises oil andmagnetic nanoparticles. A pressure of 100 mbar may be applied. Althoughthe implementation and integration of pressure-actuators is facile,magnetic manipulation of a ferro-fluid plug is likely to provide for theoptimum control of sample plug motion and reagent diffusion if performedin a looped microfluidic systems as shown in FIG. 2. Using eitherapproach we expect that the sample plug can be driven through between 20and 60 thermal cycles within a period of five minutes.

Label free intrinsic imaging according to the invention allows real timedetection of the product without the need for the inclusion ofadditional labels. Furthermore, using detectors, the amount of productcan also be quantified according to the invention.

Preferably, the methods of the invention can be carried out so that adevice with a plurality of microchannels is used.

In another aspect, the invention provides the use of the method fordetermining the presence of a nucleotide modification. A nucleotidemodification may be the substitution, deletion or addition of anucleotide or base pair. According to the invention, one or morenucleotide modifications may be detected. In particular, themodification may be STR, SNP, a Targeted Genetic Modification (GM) step.Preferably; the nucleotide modification is a SNP. In this system twoprimers are used at the 5′ end of the putative product, one of which hasa 3′ mismatch, as shown in FIG. 4. The upper wild type template has acomplementary primer, which the polymerase will be able to extend to the3′. The lower SNP containing template cannot fully anneal the primer,creating a 3′ mismatch. This will result in markedly lower product, ifindeed any. The product is detected by Label Free Intrinsic Imaging asdescribed herein.

A possible instrumentation layout for carrying out the invention isshown in FIG. 1. The light source (a Deuterium lamp—HEREAUS Noblelamp DS225/05J, optical parts (UV lenses-Newport, UV filters-Andover Optic),separation phase (Capillaries-Composite Metal Services Ltd) and detector(HAMAMATSU PDA 3904 S3904-512Q) are arrayed on a common rail. Light fromthe low-noise deuterium lamp or passes through a filter wheel allowingthe selection of detection wavelength. The light is then focused on afused silica capillary, typically with an internal diameter of 50-100micrometers (μm). As a biomolecule passes the light beam it absorbsenergy dependent on its spectral characteristics. The light beam is thenfocused on to the detector where the drop in signal due to the energyabsorbed by the biomolecule is measured.

Accordingly, in a further embodiment, the invention provides anapparatus for determining the presence of nucleic acid comprising atleast one microchannel, a ferrofluidic actuation means, heatingelements, a UV light source and a detector.

The detector is preferably a photo diode array or a charge coupleddevice.

In one embodiment, the apparatus comprises a plurality of parallelchannels which are imaged simultaneously, using an array of CCDs.

It should also be noted that the microfluidic devices can and will beintegrated with both upstream and downstream processing Components, suchas DNA extraction and product sizing. Consequently, the microfluidicplatform will be used to perform all processing tasks between sampleextraction (from the patient) to final SNP validation.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the instrumentation layout.

FIG. 2 shows a multichannel semi continuous PCR method.

FIG. 3 illustrates how a sample plug is moved repeatedly between twocontrolled heating zones to effect strand denaturation, annealing andextension in each cycle of a PCR reaction.

FIG. 4 illustrates PCR based SNP analysis.

FIG. 5 illustrates the hyperchromic effect of DNA.

FIG. 6 shows a possible product curve.

FIG. 7 shows the product from 10 PCR cycles for 300 bp denatured DNAfragment (separation in 5MDa (2.5% conc) PEO; 40 cm separation length).The top panel show single pixel data, the bottom one shows processeddata using the signal processing algorithms.

1. A method for the detection of nucleic acid the method comprising a)amplifying a nucleic acid sample wherein a solution comprising a nucleicacid sample is moved along a temperature-controlled and UV illuminatedchannel and the flow direction of the solution is altered multiple timesand b) measuring UV absorption of the nucleic acid.
 2. The method ofclaim 1 further comprising measuring the velocity of the nucleic acid.3. The method of claim 2 using velocity based signal processing.
 4. Themethod of any preceding claim comprising determining the amount ofnucleic acid present.
 5. The method of any preceding claim wherein thenucleic acid is DNA or RNA.
 6. The method of any preceding claim whereinamplification is by PCR.
 7. The method of any preceding claim whereinthe solution further comprises a PCR mixture.
 8. The method of claim 7wherein the PCR mixture comprises at least one complementary primer anda polymerase.
 9. The method of claim 8 wherein the primer is notlabelled.
 10. The method of claim 8 wherein the primer further comprisesa label.
 11. The method of claim 10 wherein the label is fluorescence.12. The method of any preceding claim wherein the temperature of thechannel is within the range of 50° C. to 100° C.
 13. The method of anypreceding claim wherein three different temperatures zones are appliedto separate parts of the channel.
 14. The method of claim 13 wherein thetemperature in the first temperature zone is within the range of 50° C.to 60° C., the temperature in the second temperature zone is about 72°C. and in the third temperature zone about 95° C.
 15. The method of anypreceding claim wherein the number of PCR cycles is 10 to
 40. 16. Themethod of claim 15 wherein the number of PCR cycles is
 20. 17. Themethod of any preceding claim wherein the amount of nucleic acid isrepresented by the detected absorption of UV light by the nucleic acidmolecules.
 18. The method of claim wherein the amount of nucleic acidproduced in a PCR cycle is represented by the detected absorption of UVlight by the nucleic acid molecules.
 19. The method of any precedingclaim wherein UV absorption is detected using a photo diode array or acharge coupled device.
 20. The method of any preceding claim wherein thesolution is moved using pressure or ferrofluidic actuation.
 21. The useof the method of any of claims 1 to 20 for the detection of a nucleicacid modification.
 22. The use of claim 21 wherein the nucleic acidmodification is SNP.
 23. An apparatus for determining the presence ofnucleic acid comprising at least one microchannel, a ferrofluidicactuation means, heating elements, a UV light source and a detector.